Histoplasma capsulatum (Hc), is a dimorphic fungal pathogen that undergoes a temperature-induced transition from a mold that grows in the soil, to a parasitic yeast form that establishes infection in the lung and is capable of causing severe systemic disease in mammals. While disease is most severe in the immunocompromised, Hc also causes serious problems in immunocompetent hosts. In mice and humans, the pathology of histoplasmosis demonstrates the pivotal role of macrophages in primary infection of the lungs, systemic dissemination, and resolution of disease. Transition from mold to yeast is the first essential step in the pathogenesis of Hc. Conidia and mycelial fragments are inhaled and germinate to the pathogenic yeast form that is responsible for all subsequent steps and interactions with host cells. Hc is a remarkably well-adapted parasite of macrophages, proliferating intracellularly in a membrane-bound compartment of relatively neutral pH. The ability to modulate and survive within the hostile environment of the macrophage is integral to Hc pathogenesis. To fully understand the course of Hc infection, we must ascertain mechanistic details regarding how Hc establishes a successful infection of macrophages, as little research has focused on identifying adhesive factors required for the initial Hc and macrophage attachment. The central goal of this project is to therefore identify adhesins employed by Hc that are required for initial macrophage attachment and subsequent virulence. Analysis of these adhesins or adhesin-related factors will reveal important insights into the molecular determinants required by intracellular parasites for macrophage entry and therefore intracellular survival. In the first aim I will generate a library of Hc insertional mutants. Mutants will be screened and enriched to yield several candidates containing mutations in genes required for macrophage attachment. In the second aim I will identify and characterize the molecular determinants that are critical for adhering to macrophages and for Hc virulence. My objective is to map the sites of insertion, resulting in low-binding yeasts, and through targeted gene disruption and complementation, verify the requirement of specific genetic elements for virulence in vitro and in vivo. In the third aim I will take a candidate gene approach to assess whether predicted adhesins, cell surface proteins, or secreted factors are required for attachment to macrophages in either the yeast or conidial phases of growth. These candidates were selected based on their transcriptional upregulation in the yeast or conidial phases of growth and/or prediction to encode cell surface or secreted factors. This approach will complement the unbiased screening approach and provide insight into whether gene products required for macrophage attachment by yeast and conidia are similar or different. The completion of this project will identify molecular determinants required for Hc attachment to macrophages and yield a fundamental understanding of the initial interaction required by Hc for the establishment of a successful infection of macrophages.